Ca2+/calmodulin disrupts AKAP79/150 interactions with KCNQ (M-Type) K+ channels

Abstract

M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K(+) transport, and are regulated by numerous receptors via G(q/11)-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, and M current is known to be regulated by Ca(2+)/calmodulin (CaM) and PIP(2), we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca(2+)/CaM and PIP(2) on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results, CHO cells stably expressing M(1) receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to "sensitize" KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose-response relations to lower concentrations. The effect on KCNQ4 was abolished by the T553A mutation of the putative PKC phosphorylation site. We then probed the role of CaM and PIP(2) in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN), CaM that cannot bind Ca(2+), to disrupt the interaction of YFP-tagged AKAP79(1-153) with CFP-tagged KCNQ2-5. Tonic depletion of PIP(2) by cotransfection of a PIP(2) phosphatase had no effect, and sudden depletion of PIP(2) did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP(2) depletion.

Figure 1.

KCNQ2-5, but not KCNQ1, interacts with AKAP79. A, Shown are images of CHO cells under TIRF illumination expressing CFP-tagged KCNQ1-5 and YFP-tagged AKAP79, using 442 or 514 nm laser lines. Images of the CFP (left, in rainbow pseudocolor) and YFP (right, in yellow pseudocolor) emissions are shown before or after YFP photobleach, as labeled. Note the brighter CFP emission (warmer colors) after YFP photobleach for KCNQ2-5, but not KCNQ1. B, Bars shows the percentage increase in CFP emission after YFP photobleach for the groups in A, as well as for the membrane-targeted CFP-YFP tandem (Rho-pYC) and for ECFP-M + AKAP79-YFP that serve as our positive and negative FRET controls, respectively. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

AKAP79 sensitizes KCNQ2/3 channels to inhibition by muscarinic receptor stimulation. Plotted are normalized KCNQ2/3 currents during the experiment from stably M₁-expressing CHO cells transfected with KCNQ2 + 3 and GFP only (A) or together with GFP-tagged AKAP79 (B), while a range of concentrations of oxo-M or 10 μm XE991 were bath applied, as indicated by the bars. Representative current traces are shown in the insets, using the indicated voltage protocol. C, Plotted are the summarized dose–response relation of the KCNQ2/3 current versus oxo-M concentration for cells transfected with KCNQ2 + 3 and GFP only (control) or together with GFP-tagged AKAP79. The data were fit by Hill equation curves, with the parameters given in the text.

Figure 3.

AKAP79 sensitizes KCNQ2-5, but not KCNQ1, channels to inhibition by muscarinic receptor stimulation. Plotted are the summarized inhibition of the current versus oxo-M concentration for M₁-expressing CHO cells transfected with KCNQ1 (A), KCNQ2 (B), KCNQ3 (A315T) (C), KCNQ4 (WT or the T553A mutant) (D), or KCNQ5 (E), either with GFP only (control, circles) or together with GFP-tagged AKAP79 (AKAP79, triangles). The data were fit by Hill equation curves, with the parameters given in the text. For all, the dotted or solid curves are the fits to the control or AKAP79 data, respectively, and in D, the gray symbols and curves represent the data from the KCNQ4 (T553A) mutant.

Figure 4.

Functional Ca²⁺/CaM, but not PIP₂ depletion, disrupts the interaction between KCNQ2-5 channels and AKAP79_1-153. Shown are images of CHO cells under TIRF illumination expressing YFP-tagged AKAP79_1-153 and CFP-tagged KCNQ2 (Q2, WT or the R345E mutant) (A), KCNQ3 WT (Q3) (B), KCNQ4 (Q4) (C), or KCNQ5 (Q5) (D), either alone or together with WT CaM, dominant-negative (DN) CaM or a PIP₂ phosphatase (PIP₂ phos), using 442 or 514 nm laser lines. Images of the CFP (rainbow pseudocolor) and YFP (yellow pseudocolor) emissions are shown before or after YFP photobleach, as labeled.

Figure 5.

Summarized data for experiments involving AKAP79_1-153. Bars are summarized data for the groups of cells shown in Figure 4, as well as the FRET between CFP-tagged KCNQ2 and YFP-tagged AKAP79_1-153, or for Rho-pYC, measured using the sensitized-emission method.

Figure 6.

AKAP79/150 localization to the membrane is not dynamically altered by depletion of PIP₂. A, Shown are conventional confocal images of CHO cells transfected with EGFP and AKAP150, fixed, and immunostained with anti-AKAP150 primary and anti-mouse Rhodamine Red secondary antibodies. Images were acquired either of GFP (using the 488 nm laser line), of Rhodamine Red (using the 543 laser line), or the merged image. B, Shown are “swept-field” confocal images of stably expressing M₁ receptor CHO cells transfected with GFP-tagged AKAP79 or YFP-PH-PLCδPH, before or after bath application of oxo-M (10 μm). The images were analyzed either as a line scan or by quantifying the fluorescence in a cytoplasmic area (as shown). Shown in the insets are the results of the line scans for the presented images. Bars show summarized cytoplasmic F/F₀ data for all cells studied, where F₀ is the initial fluorescence. C, Shown are “swept-field” confocal images of CHO cells transfected with the CFP-tagged FRB construct and untagged FKBP12 fused with a PIP₂ phosphatase, together with either GFP-tagged AKAP79 or YFP-PH-PLCδPH, before or after bath application of rapamycin (500 nm). The images were analyzed either as a line scan or by quantifying the fluorescence in a cytoplasmic area (as shown). Shown in the insets are the results of the line scans for the presented images. D, Bars show summarized cytoplasmic F/F₀ data for all cells studied, where F₀ is the initial fluorescence.

Figure 7.

Functional, but not DN, CaM disrupts the AKAP79-mediated sensitization of KCNQ2/3 channels to inhibition by muscarinic receptor stimulation. Plotted are normalized KCNQ2/3 currents during the experiments from stably M₁-expressing CHO cells transfected with KCNQ2 + 3 and GFP only (A), KCNQ2 + 3 with GFP-tagged AKAP79 (B), KCNQ2 + 3 with GFP-tagged AKAP79 and WT CaM (C), or KCNQ2 + 3 with GFP-tagged AKAP79 and DN CaM (D), while oxo-M was bath applied at concentrations of 0.2 μm or 0.8 μm, as indicated by the bars. Representative current traces are shown in the insets, using the voltage protocol as in Figure 2. E, Bars are summarized data for the groups of cells as in A–D. **p < 0.01.